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Draft by Dr. V.K.

Mishra, DPMC, Dehradun

Enzyme Electrode: Enzyme based biosensorT What is a biosensor Various definitions and terminologies are used depending on the field of application. Biosensors are known as: immunosensors, optrodes, chemical canaries, resonant mirrors,glucometers, biochips, biocomputers, and so on. A commonly cited definition is: “a biosensor is a chemical sensing device in which a biologically derived recognition entity is coupled to a transducer, to allow the quantitative development of some complex biochemical parameter”. “A biosensor is an analytical device incorporating a deliberate and intimate combination of a specific biological element (that creates a recognition event) and a physical element (that transduces the recognition event)”. The name “biosensor” signifies that the device is a combination of two parts: (i) a bio-element, and ii) a sensor-element. The basic concepts of a biosensor’s operation can be illustrated with the figure1.

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Elements of Biosensors : A biosensor essentially comprise of the following two major parts, namely : (a) Biological component –i.e., for sensing the presence as well as concentration of an analyte. The bioelement may be an enzyme, antibody, living cells, tissue, etc., and (b) Transducer device — i.e., an asembly that actually converts the biochemical signal into the corresponding electrical signal which may be adequately amplified and read finally either on a digital panel or recorded on a suitable recording device .

Draft by Dr. V.K. Mishra, DPMC, Dehradun

The schematic outline of a biosensor exhibiting the various integral components associated with it is given in fig.2

Electrochemical Electrode or Enzyme Electrode Electrochemical electrodes (or Enzyme eletrodes) are a new type of detector or biosensor that have been exclusively designed for the potentiometric or amperometric assay of substrates, for instance : alcohol, amino acids, glucose, and lactic acid. The enzyme electrode is a combination of any electrochemical probe (amperometric, potentiometric or conductimetric ) with a thin layer (10 - 200mm) of immobilised enzyme. History Enzyme electrodes are a type of biosensor that have enzyme as a biological component. The history of biosensors started in the year 1962 with the development of amperometric enzyme electrode for glucose by the scientist Leland C. Clark. The year 1969 marks first potentiometric biosensor:urease immobilized on an ammonia electrode to detect urea.During the year 1972-75, first commercial glucose biosensor

Draft by Dr. V.K. Mishra, DPMC, Dehradun

was developed by yellow spring instruments. Since then, several biosensors including enzyme electrodes were developed. Design The electrochemical electrode(enzyme electrode) is composed of a electrochemical sensor which in close contact with a thin-permeable enzyme membrane. The embedded enzymes located in the membrane produce products, such as H + ions, oxygen (O2), NH4+ ions, carbon dioxide (CO2) or ever other small molecules depending solely on the enzymatic reactions , that are rapidly detected by the particular sensor . The magnitude of the response gives the precise estimations of the prevailing concentration of the substrate.

The biological component present in a biosensor may invariably be an enzyme or a multi-enzyme system, that could also be an antibody or organelle or microbial cell or even whole slices of tissue. In these devices, enzyme is combined with electrochemical probe .The out put of device would be in terms of current (amperometric), voltage (potentiometric) or conductivity (conductimetric).) Types of Enzyme Electodes 1. Amperometric Enzyme Electrode 2. Potentiometric Enzyme Electrode 3. Conductimetric Enzyme Electrode

Draft by Dr. V.K. Mishra, DPMC, Dehradun

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The anylate is sensed by the immobilized enzyme. Following this, the progress of the enzyme reaction is monitored by the rate of formation of product or the disappearance of a reactant. If either the product or reactant are electroactive, then the progress of the reaction can be monitored as out put in form of current or potential or conductivity. I. Systems based on oxygen or peroxide electrochemistry

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The most commonly used enzymes in the design of enzyme electrodes contain redox groups which change redox state during the biochemical reaction.

Enzymes: Enzyme electrode with redox group of this type are the oxidases and the pyrroloqui noline quinone (PQQ) dependent dehydrogenases.

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In nature, oxidase enzymes such as glucose lactate and cholesterol oxidase act by oxidising their substrates, accepting electrons in the process and thereby changing to an inactivated reduced state. These enzymes are normally returned to their active oxidized state by transferring these electrons to molecular oxygen, resulting in the production of hydrogen peroxide (H2O2).  Because both oxygen and hydrogen peroxide are both electrochemically active, the progress of the biochemical reaction can be followed by either reducing the oxygen (co-substrate) or oxidising the hydrogen peroxide (product). The method based upon oxygen reduction at an O2 electrode or measurements based upon hydrogen peroxide oxidation, is indeed represents by far the most popular approach. Glucose Biosensors The most commercially successful biosensors are amperometric glucose biosensors. These biosensors have been made available in the market in various shapes and forms such as glucose pens, glucose displays, etc. The first historic experiment that served as theFig. The Clark experiment origin of glucose biosensors was carried out by Leland

Draft by Dr. V.K. Mishra, DPMC, Dehradun

C. Clark. He used platinum (Pt) electrodes to detect oxygen. The enzyme glucose oxidase (GOD) was placed very close to the surface of platinum by physically trapping it against the electrodes with a piece of dialysis membrane. The enzyme activity changes depending on the surrounding oxygen concentration. Fig. 7 shows the reaction catalyzed by GOD. Glucose reacts with glucose oxidase (GOD) to form gluconic acid while producing two electrons and two protons, thus reducing GOD. The reduced GOD, surrounding oxygen, electrons and protons (produced above) react to form hydrogen peroxide and oxidized GOD (the original form). This GOD can again react with more glucose. The higher the glucose content, more oxygen is consumed. On the other hand, lower glucose content results in more hydrogen peroxide. Hence, either the consumption of oxygen or the production of hydrogen peroxide can be detected by the help of platinum electrodes and this can serve as a measure for glucose concentration.

Mediated systems A major limitation of the peroxide system described above, is the high operating voltage (circa 0.8 volts vs the Ag/AgCl reference lectrode) required to oxidise the hydrogen peroxide resulting in the possibility of interference. The use of mediators (molecules which can shuttle electrons between the redox centre of the enzyme and the electrode) can minimise this problem as they can, depending on the compound used, be regenerated at potentials where interference from species such as ascorbate, urate and paracetamol. A vast number of compounds are capable of acting as enzyme mediators and the groups which are most frequently used in the construction of enzyme electrodes are detailed below. Of these, mediators based on metal complex Bi-enzyme systems Recently, work has focused on the direct electrical communication between an enzyme and the electrode. Although success in this field has been limited, one enzyme which has achieved this goal is horseradish peroxidase (HRP). HRP catalyses the reduction of hydrogen peroxide at the expense of a number of organic reducing compounds.

Draft by Dr. V.K. Mishra, DPMC, Dehradun

When the enzyme is linked electrically to an electrode however, the need for the organic reductant is obviated since the electrode itself provides the reducing equivalents.